The transposon Tn554, which specifies resistances to eyrthromycin (MLS) and spectinomycin in Staphylococcus aureus, is 6.7 kb in length and lacks any observable inverted repeat sequences; it transposes to a unique chromosomal ("primary") insertion site, while insertions into other sites, such as on plasmids, are only very rarely obtained. Transposition is strongly inhibited if the cell already contains a copy of Tn554 inserted in the chromosome, whereas it proceeds in a manner akin to zygotic induction upon transfer to a cell lacking a resident copy. Tn554 specifies an inhibitor, designated tnpC, that maps at the extreme left end of the transposon and which, when cloned on a plasmid vector, can act in trans to prevent the transposition of an intact copy of Tn554 to a vacant chromosomal insertion site. The mechanism of transposition of Tn554 will be studied in vivo by analysis of the intermediates that can be isolated following synchronous induction of tranposition from a high frequency donor of Tn554 into a cloned copy of the primary chromosomal insertion site. Tn554 functions involved in transposition and its regulation will be analyzed by physical and genetic characterization of mutants generated in vitro by random insertion of oligonucleotides. It is expected that mutants affecting the regulation and specificity of transposition, as well as those affecting the transposition process itself, will be obtained. Once the functions involved in transposition and the primary intermediates are known, an attempt will be made to study transposition in an in vitro system.